Human Th2 specific protein

ABSTRACT

The present invention provides for specifying the condition and type of immune-related diseases on the basis of the knowledge about the polarization of the distribution of helper T-cell subsets Th1 and Th2. More sepcifically, in this invention, the gene (B19) specific only the human Th2 is prepared and specified by a subtraction method, and a recombinant vector into which the gene is incorporated, a transformant transformed by the recombinant vector, a human-Th2-specific protein which the gene encodes and which derives from the transformant, and a monoclonal antibody against the Th2-specific protein are produced and the gene, protein, antibody, etc. are used as the means for specifying or correcting the polarization of the distribution of Th1 and Th2 to solve the above object.

TECHNICAL FIELD

The present invention relates to a Th2-specific-protein and a geneencoding the protein, and transformants, recombinant vectors andmonoclonal antibodies related to the gene.

More specifically, the present invention relates to a protein which isspecific solely for Type 2 helper T cells and which can be used as meansfor promptly and simply specifying variations in balance among helper Tcell subsets intimately involved in the occurence of atopic diseases,the progression of AIDS, etc., and also relates to a gene which encodesthis protein.

Further, the present invention relates to a recombinant vector whichharbors the gene and is used for expressing it, as well as to atransformant which is transformed with the recombinant vector.

Furthermore, the present invention relates to a monoclonal antibodyagainst the Th2-specific protein, and a hybridoma which produces themonoclonal antibody.

BACKGROUND ART

Immunology has made a remarkable progress in recent years and has addeda great contribution to the field of medicine.

Studies of immunology have revealed that cytokines produced bymacrophages, lymphocytes, etc. play the central role in promoting orsuppressing every immunological reaction, such as infection immunity,tumor immunity, allergies, or anaphylaxis.

Mosmann and Coffman, et al. classified CD4⁺ T-cell clones which areestablished from mouse spleen cells and can be cultured for a longperiod of time into two different types of subsets according to thedifference between cytokines produced by the clones (Mosmann, T. R., etal., J. Immunol.,136, 2348 (1986)).

Specifically, they classified CD4⁺ T-cell clones into the "T-helper 2(Th2) subset" and the "T-helper 1 (Th1) subset": the former principallyproduces IL(interleukin)-4, IL-5, IL-6, IL-10 and IL-13; and the latterprincipally produces IL-2, IFN (interferon)-γ, and TNF (tumor necrosisfactor)-β.

Although the existence of such subsets of helper T cells in humans wasinitially deemed dubious, it is now well accepted (Romagnani, S.,Immunology Today 12, 256 (1991), etc.).

Nowadays, the nature and functions of the helper T-cell subsets Th2 andTh1 in mice or humans are becoming much more evident. In terms ofbiological significance, they have become of great interest as dominantcells which control different immunological reactions.

In many infectious diseases or immunological diseases, polarization isobserved in the distribution of the Th1/Th2 subsets of lymphocytes ofpatients; i.e., an extreme bias arises in the distribution toward eitherone of the subset Th1 or Th2. Therefore, it is suggested that the natureof this polarization phenomenon may reflect the condition and type ofthe disease.

For example, the following are currently becoming distinct:

(1) In the case of Mycobacterium diseases, if immunological reactionswith respect to Mycobacterium are mainly delayed-type hypersensitivity(DTH) reactions, the Th1 subset is dominant, whereas if theimmunological reactions are chronic and progressive, the Th2 subset isdominant. (2) In the case of HIV diseases, the production of Th1-typecytokines is observed among many long-term nonprogressive HIV-infectedpatients. If polarization arises toward the Th2 subset, the symptoms ofthe diseases become progressive or fulminated. (3) With regard topatients having atopic diseases, if there arises polarization toward theTh2 subset, the diseases become aggravated.

In view of the foregoing, the problems to be solved by the presentinvention is to provide means for specifying the condition and type ofimmune-related diseases on the basis of the knowledge about thepolarization of the distribution of Th1/Th2 subsets (hereinafterreferred to as Th1/Th2 imbalance).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an autoradiogram showing the results of Northern blot of theclone B19;

FIG. 2 is an autoradiogram showing an in vitro translation product ofthe Th2 (B19) gene of the present invention;

FIG. 3 is an autoradiogram showing the results of Northern blot whichindicates the tissue specificity of expression of mRNA derived from theTh2 (B19) gene of the present invention; and

FIG. 4 shows the analyses with a flow cytometer after staining the Th1clone and Th2 clone cells by a membrane fluorescent antibody techniquewith the monoclonal antibody of the present invention.

DISCLOSURE OF INVENTION

The inventors of the present invention carried out extensive studies onthe above-described problems. As a result, they found that if a proteinspecific for Th2 cells, a gene which encodes the Th2-specific protein, aprotein specific for Th1 cells, and a gene which encodes theTh1-specific protein can be identified and prepared, respectively, itbecomes possible to provide means for specifying the condition and typeof an immunity-related disease of interest through use of the proteinsand genes. The present invention was completed on the basis of thisfinding.

The present invention is directed to a protein specific for human Th2cells and a gene (B19) which encodes the human-Th2-specific protein.

More specifically, the present inventors provide the invention asfollows:

According to a first aspect of the present invention, there is provideda human-Th2-specific protein having an amino acid sequence representedby sequence ID No. 6.

According to a second aspect of the present invention, there is provideda human-Th2-specific protein which has an amino acid sequencecorresponding to an amino acid sequence represented by sequence ID No. 6partially deleted, replaced, or added, and which has substantially thesame biological activities as those of the human-Th2-specific protein.

According to a third aspect of the present invention, there is provideda human-Th2-specific gene which contains a nucleotide sequence codingfor the amino acid sequence represented by sequence ID No. 6.

According to a fourth aspect of the present invention, there is provideda human-Th2-specific gene containing a nucleotide sequence representedby sequence ID No. 5.

According to a fifth aspect of the present invention, there is provideda human-Th2-specific gene which contains a nucleotide sequencecorresponding to a nucleotide sequence represented by sequence ID No. 5partially deleted, replaced, or added; which hybridizes with DNA havingthe nucleotide sequence represented by sequence ID No. 5 under stringentconditions; and which encodes a human-Th2-specific protein havingsubstantially the same biological activities as those of ahuman-Th2-specific protein having the amino acid sequence represented bysequence ID No. 6.

According to a sixth aspect of the present invention, there is provideda recombinant vector for expressing a gene, which vector contains thehuman-Th2-specific gene as defined in any one of the aforementionedaspects.

According to a seventh aspect of the present invention, there isprovided a transformant which is transformed by the recombinant vectordefined in the sixth aspect and in which the human-Th2-specific genecontained in the recombinant vector is expressed.

According to an eighth aspect of the present invention, there isprovided a monoclonal antibody which takes as its antigenic determinantany portion of the human-Th2-specific protein defined in the aboveaspects and which does not exhibit immunoreactivity with respect tohuman-Th1-specific proteins.

According to a ninth aspect of the present invention, there is provideda hybridoma which produces the monoclonal antibody defined in the eighthaspect.

The following will explain the mode for carring out the presentinvention.

As described hereinabove, a human Th2 from which human-Th2-specific geneused herein is derived is one of the subsets of human helper T cells.The human-Th2-specific gene will be hereinafter referred to as a Th2(B19) gene. Unless otherwise specified, the Th2 (B19) gene includes analtered human-Th2 (B19)-specific gene (which will be described later)that falls within the scope of the present invention.

The human Th2 is a subset of helper T cells having the followingproperties:

(1) The human Th2 cells produce IL-4 and IL-5 but produces neither IFN-γnor TNF-β.

(2) The human Th2 cells proliferate in response to IL-2 and IL-4, andthe induction of the human Th2 cells are suppressed by IFN-γ. Althoughthe other subset (i.e., the human Th1 subset) proliferates similarly inresponse to IL-2 (as well as with IL-12), the induction of the human Th1cells are suppressed by IL-4 as contrasted with the human Th2 cells.

(3) Surface markers for human Th2 cells which can definitely distinguishTh2 cells from Th1 cells are not yet found. The human Th2 cells, likethe human Th2 cells, have phenotypes of CD44^(bright), CD45RB^(dull),and LECAM-1^(dull).

(4) The human Th2 cells encourage antibody production and induces,particularly, IgE production.

(5) The human Th2 cells promote the differentiation and proliferation ofmast cells and eosinophils.

(6) The human Th2 cells do not induce antigen-specific DTH reaction andbecome dominant if immunological reactions are chronic and progressive.

The Th2 (B19) gene of the present invention can be obtained byestablishing human Th2 clones having the above-described characteristicsand producing a cDNA library of the human Th2 cells from thethus-established human Th2 clones.

A. Establishment of the Human Th2 Clones:

As a preceding step for establishing desired human Th2 clones, a CD4⁺T-cell population which is known to contain these clones is produced.

The CD4⁺ T cell population can be produced according to a conventionalmethod disclosed, for example, in "Gianfranco, F. D. P., et al., J.Clin. Invest. 88, 346 (1991)."

More specifically, for example, peripheral blood mononuclear cells maybe separated from the whole blood of humans, and the thus-separatedperipheral blood mononuclear cells may be stimulated by variousT-cell-activators to thereby produce a desired CD4⁺ T-cell population.The T-cell-activators may include; e.g., non-specific T-cell-activatorssuch as kidney bean-derived phytohemagglutinin (PHA); cytokines such asIL-2, IL-4, or IL-12; or stimulus antigens such as PPD or mite extracts.

Prior to isolation of CD4⁺ T cell clones, which will be described below,the CD4⁺ T cell population is preferably subjected to removal ofelements other than the CD4⁺ T cells; e.g., CD8⁺ T cells. For example,there can be employed a method for concentrating only the CD4⁺ T cellsthrough use of magnetic beads coupled to anti-CD4 antibody.

After completion of the above-described induction process, the CD4⁺T-cell clones are isolated. They can be isolated according to methodsknown per se such as the limiting dilution technique.

More specifically, for example, cells are seeded in a medium including aPHA and IL-2 on a 96-well microplate so as to be 0.5 to 10 cells/well.The medium is replaced with a fresh IL-2-added medium every three tofour days. The surface marker of the cells which have been ascertainedto proliferate (normally within two to four weeks) is examined. CD4⁺T-cell clones can be isolated by selecting only the CD4-positive clones.

From among the thus-isolated CD4⁺ T-cell clones, human Th2 clones ofinterest can be selected.

The human Th2 clones are selected from the CD4⁺ T-cell clones accordingthe known differences in properties between the human Th2 and human Th1cells.

Specifically, for example, clones which produce IL-4 in response to thestimulation of anti-CD3 antibody but do not produce IFN-γ can beselected as the human Th2 clones. (In contrast, clones which produceIFN-γ but do not produce IL-4 are selected as the human Th1 clones.)

B. Preparation of Human-Th2-specific cDNA:

With regard to the cDNA of the human Th2 and that of the human Th1, itis anticipated that there exist nucleotide sequences common to bothtypes of cDNA and nucleotide sequences specific for the respectivetypes.

To prepare cDNA specific for the human Th2 cells of interest on thebasis of such an anticipation, it is advantageous to use a so-calledsubtraction method by which cDNA species common to the human Th1 andhuman Th2 cells are eliminated from cDNA molecules of the human Th2cells.

The subtraction method may include, e.g., a method reported by Davis etal., (Davis, M. M. et al., Proc. Natl. Acad. Sci. U.S.A., 81, 2194(1984)).

This method is intended to concentrate cDNA clones specific for one oftwo cell types, through hybridization of the cDNA to be subtracted witha greatly excessive amount of poly (A)⁺ RNA derived from the other celltype, and the screening of the cDNA library through use ofnon-hybridized residual cDNA as a probe.

Since this method requires a large amount of poly (A)⁺ RNA, the methodhas a disadvantage of being difficult to carry out the method if a largequantity of poly (A)⁺ RNA is not readily available.

To overcome this problem, another method has already been reported whichuses the PCR technique in order to carry out subtraction through use ofa comparatively small amount of poly (A)⁺ RNA as a starting material[e.g., a gene expression screen method (Wang, Z. and Brown, D. D., Proc.Natl. Acad. Sci. U.S.A.,88, 11505 (1991), etc.]. This method ischaracterized by the amplification of cDNA serving as a startingmaterial by use of the PCR method. This method also has the advantage ofbeing capable of cloning a very small amount of mRNA through repetitionof the subtraction operation and the amplifying operation by PCR.

In general, culturing a large amount of normal CD4⁺ T-cell clones andsecuring a large amount of mRNA which serves as a template of cDNA isdifficult. For this reason, the present invention preferably employs the"gene expression screen method," for example, from among theaforementioned subtraction methods.

More specifically, cDNAs derived from human Th2 clones and human Th1clones are prepared by generally known methods (e.g., a method in whicha reverse transcriptase is used, with poly (A)⁺ RNA being employed as atemplate of cDNA), and the resultant cDNAs are amplified by PCR.

In amplifying cDNA, it is desirable to previously subject the cDNA totreatment with restriction enzymes or ultrasonic treatment so as toobtain cDNA fragments which are suitable in length for amplificationaccording to the PCR method.

For example, specific primers which contain different nucleotidesequences respectively for the human Th1 and the human Th2 cells may beused as the PCR primers needed to amplify cDNA according to the PCRmethod. Usually, these specific primers are prepared by chemicalsynthesis. Since only the human-Th2-derived cDNA is amplified throughuse of a specific primer after the subtraction, this method has theadvantage of minimizing the amplification of a trace amount ofhuman-Th1-derived cDNA which may contaminate the human-Th2-derived cDNA.

In this case, it is necessary to previously connect linker--whichcontain sequences capable of being annealed with the PCR prime--to bothends of the cDNA fragments. Therefore, in the aforementionedfragmentation, it is desirable to use a restriction enzyme whichproduces cDNA fragments having terminals capable of being linked withthe linkers.

After the cDNA fragments derived from the human Th1 and Th2 clones havebeen attached to the PCR linkers, fragments having a certain length areselected from among the cDNA fragments by suitable fractionation methodssuch as agarose gel electrophoresis. The thus-chosen cDNA fragments areamplified by the PCR method, whereby the amplified cDNA fragments can beused as a starting material for subtraction.

With the thus-prepared cDNA fragments, a gene library containing thehuman Th2 gene of interest according to the present invention can beprepared by subtracting the cDNA fragments which have the nucleotidesequences common to the human Th1 and Th2 from thehuman-Th2-clone-derived cDNA fragments.

This selection may be performed by hybridizing a given quantity ofhuman-Th2-clone-derived cDNA fragments with an excessive quantity oflabeled cDNA fragments which are derived from the human Th1 clones. ThecDNA hybridized with the human-Th1-clone-derived cDNA fragments can beeliminated according to the labels, and the remaining cDNA fragments canbe handled as cDNA fragments based on the nucleotide sequence specificsolely for the human Th2 cells.

The labels used herein are not limited to any particular labels, so longas they allow the use of the foregoing screening method. Needless tosay, it is preferable to use means which enables easy labeling and easyremoval of the label. In this respect, it is preferable to use a methodin which cDNA fragments are labeled, for example, with biotin, and theresultant labeled cDNA fragments are caused to be adsorbed ontostreptavidin.

The thus-screened cDNA fragments based on the nucleotide sequencespecific only for the human Th2 are amplified again by the PCR methodand then screened by the foregoing screening means. Through repetitionof these processes, the cDNA fragments of interest can be concentratedand amplified.

A gene library containing the Th2 (B19) gene of the present inventioncan be obtained through use of the thus-prepared cDNA fragments.

The gene library can be prepared through use of a method known per se.

Briefly, the cDNA fragments are inserted into a suitable vector used fortransferring a gene, and the vector is introduced into a hostcorresponding to the vector, thereby enabling preparation of a genelibrary of interest. Here, it is possible to check whether or not thecDNA fragments have been inserted into the vector, for example, by meansof the color selection based on the activity of a lac Z gene, forexample, in the vector.

The vector for introduction purposes is not particularly limited. Forexample, useful plasmids include pBluescript, pUC18, pBR322, pBGP120,pPCφ1, pPCφ2, pPCφ3, pMC1403, pLG200, pLG300, pLG400, etc; useful λphages include λ gt10, or λ ZAPII, etc. In consideration of ease ofhandling, a plasmid which contains the lac Z gene as a selection markeris preferably used. More specifically, of the above-described vectors,pBluescript, pUC18, or pBGP120 is preferably used.

The gene library may alternatively be prepared through use of acommercially available gene library preparation kit.

C. Isolation of the Th2 (B19) Gene of the Present Invention:

It is possible to directly extract DNA from the gene library prepared inthe above-described manner and then to determine the sequences of someof the DNA to screen the clones containing the present Th2 (B19) genefrom the sequences, but it is desirable to further screen the clonescontaining the present Th2 (B19) gene in advance to certainly specifythe same.

As a screening method, a generally known screening method may be used.For example, there are first prepared genes derived from a gene librarywhich has been prepared in the previous-described manner and is based ona human-Th2-specific genes, and genes derived from a gene library whichhas been prepared separately and is based on a human-Th1-specific genes.These genes are labeled to form labeled probes, and the labeled genesare then hybridized with a replica of the gene library based on ahuman-Th2-specific genes. Subsequently, clones which hybridize with aprobe of the human-Th2-specific genes but does not hybridize with aprobe of the human-Th1-specific genes are selected. The selected clonescan be used for determining the nucleotide sequence of the Th2 (B19)gene of the present invention, which will be described later, as theclones which harbor the Th2 (B19) gene of the present invention.

To ensure extra care, there may be determined clones to be used fordetermination of the nucleotide sequence of the Th2 (B19) gene, whichwill be described later, for example, by comparison of expressionpatterns of mRNA through use of the Northern blotting technique in whichthe total RNA or poly (A)⁺ RNA of the human Th2 cells and the human Th1cells are used.

The nucleotide sequence of the Th2 (B19) gene of the present inventioncontained in the thus-prepared clones can be in general determinedthrough use of a known method.

The nucleotide sequence of the Th2 (B19) gene of interest can bedetermined through use of, for example, a Maxam-Gilbert method (Maxam,A. M., and Gilbert, W., Proc. Natl. Acad. Sci. U.S.A., 74, 560 (1977)),a genomic sequence method (Church, G. M. and Gilbert, W., Proc. Natl.Acad. Sci. U.S.A., 81, 1991 (1984)), a multiplex method (Church, G. M.,and Kieffer-Higgins, S., Science, 240, 185 (1988)), a cycle sequencemethod (Murray, V., Nucleic Acids Res.,17, 8889 (1989)), or a dideoxymethod (Sanger, F., et al., Proc. Natl. Acad. Sci. U.S.A., 74, 5463(1977)).

Alternatively, as a matter of course, the nucleotide sequence may bedetermined through use of a nucleotide sequence automatic analyzer towhich the principles of the foregoing methods are applied.

On the basis of the thus-determined nucleotide sequence of the Th2 (B19)gene, the Th2 (B19) gene itself can be obtained.

More specifically, provided that the cDNA of the human Th2 cells whichserves as the source of the Th2 (B19) gene prepared in theabove-described manner is used as a template, and that the DNA fragmentcontaining the sequences on the 5'-terminal site and the 3'-terminalsite of the Th2 (B19) gene determined in the previously-described manneris used as a primer, the Th2 (B19) gene can be amplified in largequantity by the aforementioned PCR method and obtained.

Alternatively, a traditional method is also available in which the fulllength of the Th2 (B19) gene of the present invention is obtained byselecting a clone containing the Th2 (B19) gene from the DNA genelibrary prepared from the human Th2 cells, with a human Th2 genefragment itself whose nucleotide sequence has been determined beingemployed as a probe.

Further, the Th2 (B19) gene of the present invention can be produced bychemical synthesis through use of a generally known method such as aphosphite-triester method (Ikehara, M., et al., Proc. Natl. Acad. Sci.U.S.A.,81, 5956 (1984)). In addition, the Th2 (B19) gene can besynthesized through use of a DNA synthesizer to which the chemicalsynthesis method is applied.

The inventors of the present invention have realized the existence ofaltered genes containing the nucleotide sequence of the Th2 (B19) genewhose nucleotides are partly deleted, replaced, or added by alteration(these altered genes have a homology of about 70% or more with respectto the intact Th2 (B19) gene). Such altered genes also fall within thescope of the present invention.

A human Th2 gene which can fall within the scope of the presentinvention is a human-Th2-specific gene which hybridizes with DNAcontaining a nucleotide sequence represented by sequence ID No. 5 (whichwill be described later) under stringent conditions or conditions underwhich a hybrid between DNA is less apt to be formed in a system. Morespecifically, the conditions include the temperature of the system (thehigher the temperature, the lower the likelihood of the hybrid beingformed), the concentration of salt (the lower the salt concentration,the lower the likelihood of the hybrid being formed), and theconcentration of a denaturing agent such as formamide (the higher theconcentration of the denaturing agent, the lower the likelihood of thehybrid being formed). Further, the human-Th2-specific gene encodes ahuman-Th2 (B19)-specific protein which has substantially the identicalbiological activities as those of a human-Th2-specific proteincontaining an amino acid sequence represented by sequence ID No. 6(which will be described later).

The term "substantially identical" used herein signifies that withregard to biological activities the human-Th2 (B19)-specific protein isqualitatively and/or quantitatively identical to human-Th2(B19)-specific protein for comparison.

Specific biological activities of the human-Th2 (B19)-specific proteinwill be described later.

A gene can be altered in a desired way through use of a generally knownmethod; e.g., a so-called site-specific mutagenesis (Mark, D. F., etal., Proc. Natl. Acad. Sci. U.S.A., 81, 5662 (1984)).

A local Th1/Th2 balance of a disease can be checked through use of theTh2 (B19) gene of the present invention produced in the above-describedmanner.

Specifically, a local Th1/Th2 balance of a disease can be checked byextracting mRNA from local tissue of the disease, and measuring thelevel of expression of the Th2 (B19) gene in the tissue through use of;e.g., an RT-PCR method ("PCR Protocols, A Guide to Methods andApplications" Innis, M. A., et al., ed., Academic Press, San Diego,1990).

As mentioned herein under the heading "Background Art", the checking ofthe Th1/Th2 balance allows more reliable ascertainment of variations inthe symptoms of diseases to which a Th1/Th2 imbalance is of importance;e.g., HIV diseases, allergic diseases, or various infectious diseases.

Although, as a matter of course, the Th1/Th2 balance can be checkedthrough use of polyclonal/monoclonal antibodies specific to human Th2cells, which will be described later, the foregoing checking means iseffective in the case where the use of these antibodies is difficult;e.g., the case where the quantity of expression of the target protein isa trace amount.

D. Manufacture of the Human-Th2 (B19)-specific Protein of the PresentInvention:

Through use of the thus-produced Th2 (B19) gene of the presentinvention, a recombinant human-Th2-specific protein (hereinafterreferred to as a "human Th2 (B19) protein") can be manufactured. Unlessotherwise specified, the human Th2 (B19) protein includeshuman-Th2-specific proteins which can be translated from theabove-described altered genes. As a matter of course, these alteredproteins which can be translated from the altered genes havesubstantially the identical biological activities as those of theunaltered human Th2 (B19) protein of the present invention.

The human Th2 (B19) protein of the present invention can be manufacturedaccording to a generally known conventional gene recombination techniqueby use of the Th2 (B19) gene of the present invention.

More specifically, the Th2 (B19) gene of the present invention isinserted into a gene expression vector which is in such a form as to beable to express the Th2 (B19) gene of the present invention. Therecombinant vector is transferred into a host whose propertiescorrespond to those of the vector, whereby it is transformed. The humanTh2 (B19) protein of interest can be manufactured by culturing thetransformant.

Preferably, there is used herein a gene expression vector which usuallypossesses a promoter and an enhancer in the upstream region, and atranscription terminating sequence in the downstream region of a genewhich is to be expressed.

Expression of the Th2 (B19) gene of the present invention is not limitedto a direct expression system but may be accomplished in; e.g., a fusedprotein expression system which utilizes a β-galactosidase gene, aglutathione-S-transferase-gene, or a thioredoxin gene.

Examples of gene expression vectors include vectors whose hosts are E.coli, e.g., pQE, pGEX, pT7-7, pMAL, pTrxFus, pET, or pNT26CII; vectorswhose hosts are Bacillus subtilis, e.g. pPL608, pNC3, pSM23, or pKH80;vectors whose hosts are yeast, e.g., pGT5, pDB248X, pART1, pREP1, YEp13,YRp7, or YCp 50; and vectors whose hosts are mammal cells or insectcells, e.g., p91023, pCDM8, pcDL-SRα 296, pBCMGSNeo, pSV2dhfr, pSVdhfr,pAc373, pAcYM1, pRc/CMV, pREP4, or pcDNAI.

These gene expression vectors may be selected in accordance with thepurpose of expression of the human Th2 (B19) protein of the presentinvention. For instance, in a case where the human Th2 (B19) protein isintended to be expressed in large quantity, it is desirable to select agene expression vector capable of choosing E. coli, Bacillus subtilis,or yeast as its host. In contrast, in a case where the human Th2 (B19)protein of the present invention is intended to be expressed even in asmall amount so as to become active reliably, a gene expression vectorcapable of choosing mammal or insect cells as its host is preferablyselected.

Although the existing gene expression vector can be selected asmentioned above, as a matter of course, a gene expression vector mayalternatively be prepared according to the purpose of expression asrequired.

These recombinant vectors also fall within the scope of the presentinvention.

The transfer of the vector harboring the Th2 (B19) gene into a host celland associated transformation can be carried out by means of a commonlyemployed method; e.g., a calcium chloride method or an electroporationmethod for the case of the vectors which choose E. coli or Bacillussubtilis as their host cell; or a calcium phosphate method, anelectroporation method, or a liposome injection method for the case ofvectors which choose mammal or insect cells as their host cell.

The human Th2 (B19) protein of interest is accumulated by culturing thethus-obtained transformants according to a conventional method (theabove-described transformants also fall within the scope of the presentinvention).

A medium used in the cultivation can be selected according to theproperties of the host, as required. For example, if the host is E.coli, LB or TB mediums can be used as required. Further, if the host isa mammalian cell, an RPMI1640 medium can be used, as required.

The human Th2 (B19) protein can be isolated and purified from theculture products obtained by the cultivation according to a conventionalmethod. For example, the human Th2 (B19) protein is isolated andpurified from the culture products by various processing operations,utilizing physical and/or chemical properties of the human Th2 (B19)protein of the present invention.

More specifically, the isolation and purification of the protein can beaccomplished through use of processing making use of a proteinprecipitant, ultrafiltration, gel filtration, high-performance liquidchromatography, centrifugal separation, electrophoresis, affinitychromatography using a specific antibody, or dialysis. These techniquesmay be used singly or in combination.

In this way, the human Th2 (B19) protein of the present invention can beisolated and purified.

In the Th2 (B19) gene expression system, the T cells or bone marrowcells isolated as a host from a patient can be utilized for so-calledgene therapy by transforming the cells through use of the Th2 (B19)gene, and returning the resultant transformants to the patient.

In this case, virus vectors such as retroviruses or adenoviruses may bementioned as the gene expression vectors.

The gene therapy which uses the foregoing transformants can be appliedto patients whose diseases are principally due to a Th1/Th2 imbalance inwhich Th1 is dominant. More specifically, the transformants areadministered to patients having, for example, multiple sclerosis orrheumatic arthritis. The Th1-dominant Th1/Th2 imbalance, which is theprincipal cause of these diseases, can be treated by gene therapy inwhich the administered transformants are caused to express human Th2(B19) protein within the body of the patients.

E. Manufacture of an Antibody against the Human Th2 (B19) Protein of thePresent Invention

The present invention is also directed to an antibody against the humanTh2 (B19) protein of the present invention.

The polyclonal antibody of the present invention can be manufacturedfrom immune serum derived from animals which are immunized with thehuman Th2 (B19) protein serving as an immunogen.

The human Th2 (B19) protein used herein as the immunogen is not limitedto any particular type of immunogen. As a matter of course, the humanTh2 (B19) protein that is encoded by the Th2 (B19) gene [furtherincluding a Th2 (B19) gene having a nucleotide sequence partly altered]prepared in the above-described manner, can be used as the immunogen.Further, a fragment of the human (B19) protein which a partial fragmentof the Th2 (B19) gene encodes, and a partial peptide of the human Th2(B19) protein which is obtained through direct enzyme processing of theprotein, or chemical synthesis of part of the human Th2 (B19) gene canbe also used as an immunogen during manufacture of the polyclonalantibody of the present invention.

Cell lines derived from an animal which is of the same species andgenealogy as the animal to be immunized are transformed with expressionvectors which include genes encoding the human Th2 protein [includingthe human Th2 (B19) protein of the present invention] or the partthereof. Subsequently, the thus-transformed cells are transplanted tosuitable animal to be immunized, whereby the polyclonal antibody of thepresent invention can be prepared. Specifically, the transformed cellscontinuously form the human Th2 protein within the body of the animal towhich the transformed cells have been transplanted, and an antibodyagainst the human Th2 protein is produced. This antibody may be alsoused as the polyclonal antibody of the present invention (Nemoto, T., etal., Eur. J. Immunol., 25, 3001 (1995)).

As is the case with the transplantation of the transformed cells, thepolyclonal antibody can be manufactured by direct administration to theanimal of an expression vector which expresses the human Th2 protein byintramascular or subcutaneous injection such that the human Th2 proteinis continuously produced within the animal (Raz, E., et al., Proc. Natl.Acad. Sci. U.S.A., 91, 9519 (1994)).

The monoclonal antibody of the present invention can be manufactured inthe same manner of manufacturing the polycolnal antobody of the presentinvention by the steps of producing hybridomas between myeloma cells andimmunocytes of animals that are immunized, selecting clones whichproduce human-Th2-protein-recognizable antibodies, and culturing thethus-selected clones.

The animal to be immunized is not limited to any particular kind ofanimal. Mice and rats can be widely used. However, in the case of themanufacture of the monoclonal antibody, an animal is preferably selectedin consideration of the compatibility of the animal with myeloma cellsused for cell fusion.

Immunity can be induced by a commonly-employed method; e.g., byadministering the immunogen to an animal to be immunized by intravenousinjection, intradermal injection, subcutaneous injection, orintraperitoneal injection.

More specifically, the immunogen is administered to the animal severaltimes every two to fourteen days by the above-described means, togetherwith an ordinary adjuvant as desired. As a result, immune serum usefulfor manufacturing the polyclonal antibody or immunocytes useful formanufacturing the monoclonal antibody; e.g., immunized spleen cells, canbe obtained.

In the case of manufacture of the monoclonal antibody, the following maybe used as a myeloma cell which serves as a parent cell to be fusedtogether with the immunocyte; e.g., SP2/0-Ag14, P3-NS1-1-Ag4-1,MPC11-45, 6. TG1. 7 (all of which are derived from mice); 210. RCY.Ag 1. 2. 3. (which is derived from rats); and SKO-007, and GM15006TG-A12(both of which are derived from humans).

The immunized cells can be fused with the myeloma cells by knownmethods, for example, a method reported by Kohler and Milstein [Kohler,G. and Milstein, C., Nature, 256, 495 (1975)].

Specifically, the cell fusion is carried out within an ordinary culturemedium to which promoting agents such as dimethylsulfoxide have beenadded as required in order to improve the efficiency of fusion, in thepresence of a known ordinary fusion accelerator such as polyethyleneglycol (PEG) or Sendai virus (HVJ) to thereby generate hybridomas.

A hybridoma of interest may be isolated by culturing it in an ordinarymedium for selection purposes; e.g., a HAT (Hypoxanthine, Aminopterin,Thymidine) medium. In other words, a hybridoma of interest may beisolated by culturing fused cells in the medium for a sufficient periodof time to kill cells other than the hybridoma. The thus-obtainedhybridoma can be subjected to the screening of the objective monoclonalantibody and the cloning by an ordinary limiting dilution technique(This type of hybridoma also falls within the scope of the presentinvention.).

A monoclonal antibody-producing line of interest can be screened by acommonly-employed retrieving method; e.g., an ELISA method, a plaquemethod, a spot method, agglutination, an Ouchterlony test, or a RIAmethod.

The thus-prepared hybridoma which produces ahuman-Th2-protein-recognizable monoclonal antibody of interest can besubcultured in an ordinary medium or can be stored for a long period oftime in liquid nitrogen.

A monoclonal antibody of interest is collected from the culturesupernatant by culturing the hybridoma according to a customary method.Alternatively, the monoclonal antibody is collected by administering thehybridoma to an animal having compatibility with the hybridoma so as toinduce the hybridoma to proliferate, and collecting ascites from theanimal.

Further, monoclonal antibodies of interest may be obtained by culturingimmunocytes in vitro in the presence of the human Th2 protein orportions of the protein, preparing through use of the cell fusion meanshybridomas between the immunized cells and myeloma cells after lapse ofa predetermined period, and screening the antibody-producing hybridomas(Reading, C. L., J. Immunol. Meth., 53, 261 (1982); Pardue, R. L., etal., J. Cell Biol., 96, 1149 (1983)).

Monoclonal antibodies of interest can alternatively be manufacturedthrough direct use of the Th2 (B19) gene or portions of the gene withoutuse of the human Th2 (B19) protein as an immunogen.

More specifically, it is also possible to manufacture monoclonalantibodies which specifically recognize the human Th2 (B19) protein, bydirectly immunizing an animal through use of the Th2 (B19) gene [at thistime, a gene expression recombinant vector containing the Th2 (B19) genecan be used as an immunogen], and by using immunocytes of thegene-immunized animal.

The thus-obtained polyclonal or monoclonal antibodies may be purified byordinary means such as salting out, gel filtration, or affinitychromatography.

The thus-obtained polyclonal or monoclonal antibodies exhibit specificreactivity against the human Th2 protein.

The polyclonal and monoclonal antibodies can be used as means forchecking the Th1/Th2 balance within the body. In other words, theTh1/Th2 balance in the body is checked by determining the amount ofhuman Th2 cells in a specimen through use of the antibodies inconjunction with ELISA, RIA, immunohistochemistry, flow cytometry, orthe Western blotting technique. As mentioned in the section with theheading "Background Art", it becomes possible to more reliably ascertainvariations in the symptoms of diseases to which a Th1/Th2 imbalance isof importance; e.g., atopic diseases or HIV diseases.

The thus-obtained polyclonal and monoclonal antibodies may also be usedas antibodies for correcting, for example, a Th2-dominant Th1/Th2imbalance.

With regard to antibodies derived from animals, they are acknowledged tohave antigenicity against humans if directly administered to humans.Thus, the animal-derived antibodies are not suitable for administrationto humans. For this reason, a variable region of the gene of theanimal-derived monoclonal antibody is subjected to cloning, and a geneof this variable region and a gene of the constant region in a gene of ahuman antibody are fused together. A fused antibody can be manufacturedby inducing expression of the thus-fused gene (Clarkson, T., et al.,Nature, 352, 624 (1991)).

This technique may be applied to the foregoing monoclonal antibody;namely, the fused antibody formed by fusion of the variable region ofthe animal-derived monoclonal antibody with the constant region of thehuman antibody can be also used as an antibody for correcting, forexample, the Th2-dominant Th1/Th2 imbalance.

EXAMPLES

The present invention will next be described by way of examples, whichshould not be construed as limiting the invention.

Example 1

Manufacture of a Th2 (B19) Gene of the Present Invention

(1) Preparation of Helper T-cell Clones

In order to chiefly induce human Th1 cells, 1 μg/ml of PHA (by EYlaboratories), 50 ng/ml of rIFN-γ (by Genzyme), and 5 ng/m of rIL-12 (byR&D Systems) were added to the culture of peripheral blood mononuclearcells (PBMC) (10⁶ cells/ml) isolated from a healthy human. The mixturewas cultured for five days. Independently, in order to chiefly inducehuman Th2 cells, 2% (v/v) mite extract (by Torii), 20 ng/ml of rIL-4 (byGenzyme), and 5 μg/ml of monoclonal anti-IFN-γ antibody (by Genzyme)were added to the PBMC, and the resultant mixture was cultured for fivedays.

After lapse of five days, 40 U/ml of rIL-2 (by Shionogi) was added toeach of the culture of PBMC. The PBMC were then further cultured forseven to ten days.

In order to isolate CD4⁺ T cells from the cultured cells, the culturedcells were adsorbed by magnetic beads (by Dynal) linked with anti-CD4antibodies. The cells magnetically attracted to the magnetic beads werecollected. The CD4⁺ T cells were dissociated from the magnetic beads bya magnetic bead separation reagent (by Dynal), whereby the CD4⁺ T cellswere obtained.

Next, the thus-purified CD4⁺ T cells were cultured in 15% fetal calfserum-added RPMI 1640 medium supplemented with 0.5 μg/ml of PHA and 40U/ml of rIL-2 in a 96-well microplate (0.5 cells/well). The medium wasreplaced with a fresh IL-2-added medium every three to four days. Thesurface marker of the cells which were observed to have proliferated wasexamined by immunofluorescence. Only the clones which were positive withrespect to the CD4 were selected, and the thus-selected clones weretaken as CD4⁺ T-cell clones of interest (Gianfranco, F. D. P., et al.,J. Clin. Invest.,88, 346 (1991)).

Next, in order to examine the type of each of the CD4⁺ T-cell clones,the CD4⁺ T-cell clones (6×10⁵ cells/300 μl/well) were cultured for 24hours on a 48-well plate coated with anti-CD3 antibodies (OKT3: by OrthoPharmaceutical). Concentrations of IFN-γ and IL-4 in the culturesupernatant were measured by ELISA which uses the respective monoclonalantibodies.

As a result, the clones that produced IL-4 but did not produce IFN-γwere taken as human Th2 clones. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                                   Cytokine                                                                      production                                                         Primary    (ng/ml) .sup.a)                                    Clone  Donor    Stimulation                                                                              IFN-γ                                                                          IL-4  Th type                               ______________________________________                                        1P04   KN       PHA        >50.0  <0.2  Th1                                   2P15   KT       PHA        19.2   <0.2  Th1                                   2P26   KT       PHA        <0.5    9.0  Th2                                   KND4   KN       Der .sup.b)                                                                              <0.5    4.9  Th2                                   ______________________________________                                         .sup.a)  The cells (6 × 10.sup.5  cells/300 μl/well) were            cultured for 24 hours in a 48well plate to which OKT3 had been                immobilized. Concentrations of IFNγ and IL4 in the culture              supernatant were measured by ELISA.                                           .sup.b)  Mite extract                                                    

(2) Preparation of a Subtracted cDNA Library

Poly (A)⁺ RNA was respectively prepared from the human Th2 clones (2P26)and the human Th1 clones (2P15) obtained in (1), by a customary methodmaking use of oligo dT latex (by Nippon Roche K.K.). While the poly (A)⁺RNA was used as a template, about 300 ng of cDNA was generated for eachpoly (A)⁺ RNA through use of an oligo (dT) primer (by Pharmacia) andMMLV reverse transcriptase (by Pharmacia). Next, to cut each cDNA into astrand length suitable for an amplifying process of the PCR method, thecDNA was digested for five hours at 37° C. through use of restrictionenzymes AluI (by Toyobo) (84U) and RSaI (by Toyobo) (48U), whereby eachof the following PCR linkers which differ from one another was linkedwith each cDNA [Balzer, H. J., and Baumlein, H., Nucleic Acids Res., 22,2853 (1994)]:

Linker for Human Th2:

5'-AGT TAC ACG TCT AGA ATG GCT-3' (Sequence ID No. 1)

3'-ATAG TCA ATG TGC AGA TCT TAC CGA -5' (Sequence ID No. 2)

Linker for Human Th1:

5'-CTC TTG CTT GAA TTC GGA CTA-3' (Sequence ID No. 3)

3'-ACAC GAG AAC GAA CTT AAG CCT GAT-5' (Sequence ID No. 4)

Only the cDNA fragments having the molecular-weight range from 0.2 Kbpto 2 Kbp were collected after fractionation by agarose gelelectrophoresis. The thus-obtained 2P26-derived cDNA fragments and the2P15-derived cDNA fragments were amplified by the PCR method through useof the following unique PCR primers (30 heat cycles each comprising: 94°C.×1 min.+50° C.×1 min.+72° C.×2 min.):

Primer for Human Th2:

5'-AGT TAC ACG TCT AGA ATG GCT-3' (Sequence ID No. 1)

Primer for Human Th1:

5'-CTC TTG CTT GAA TTC GGA CTA-3' (Sequence ID No. 3)

PCR products resulting from the PCR reaction were used as startingmaterials for subtraction purposes.

More specifically, an excessive amount of the PCR products (100 μg)derived from the biotin-labeled human Th1 (2P15) was added to the PCRproducts (5 μg) derived from the human Th2 (2P26). [Photoreactive biotin(100 μg) (by Vecter Laboratory) was added to DNA (100 μg). The mixturewas placed stationary about 15 cm below a 160W Sun Lamp while beingcooled on ice. The mixture was exposed to light for 15 mins. Thenon-reacted biotin was eliminated from the PCR product by butanolextraction. After repetition of these operations, biotinylated DNA wasdissolved in Tris-EDTA buffer (TE), whereby biotin-labeling of the DNAwas completed.] The resultant mixture was subjected to thermaldenaturation at 100° C., so that each PCR product was dissociated into asingle strand. The strands were hybridized with each other. Next, 100 μgof streptavidin (by Life Technologies) was added to the system, wherebycDNA derived from 2P15 which was hybridized with both free cDNA derivedfrom 2P15 and that from 2P26 was adsorbed to the streptavidin, and thecDNA was eliminated from the system by extraction withphenol-chloroform. As a result of extraction, the cDNA containing anucleotide sequence common to the cDNA derived from 2P15 was subtractedfrom the cDNA derived from 2P26, whereby subtraction for concentratingthe 2P26-specific cDNA was completed.

The thus-concentrated 2P26-specific cDNA was repeatedly subjected to PCRamplification and subtraction in the above-described manner twice. Afterthe cDNA specific for 2P26 had been concentrated further, it wassubjected to PCR amplification in the same way as described above,whereby about 3 μg of cDNA was afforded.

The thus-prepared 2P26-specific cDNA was inserted to pBluescript SK(-)(by Stratagene) and cloned, thereby completing preparation of asubtracted cDNA library. Subsequently, a portion of the subtracted cDNAlibrary was used to transform E. coli (E. coli JM 109 strain).

(3) Isolation of the Th2 (B19) Gene Fragments

The subtracted cDNA that was derived from 2P26 and obtained in step (2)and the subtracted cDNA that was derived from 2P15 and obtained in thesame way were labeled with ³² P through use of a commercially availablerandom priming labeling kit (by Takara), so that they were formed intoradioactive probes.

Independently, the E. coli that had been transformed by part of the2P26-derived cDNA library prepared in step (2) was seeded onto theplate. Two pairs of replica filters were formed with respect to coloniesgrown on the plate. These two pairs of replica filters were subjected tohybridization with the above-described two types of radioactive probes.They were washed with 0.1×SSC, and E. coli colonies containing cDNAhomologous with the cDNA present in the probes were identified byautoradiography.

As a result of the screening of about 3,400 colonies according to thismethod, there were observed 201 colonies that did not provide a positivesignal with respect to the subtracted 2P15-derived cDNA probe butprovided a positive signal with respect to the subtracted 2P26-derivedcDNA probe. With regard to these 201 cDNA clones, the difference betweenthem was examined by a colony hybridization technique, whereby 60independent clones which were not hybridized with one another wereobtained.

For these 60 clones, the difference between 2P26 and 2P15 with regard toexpression of mRNA was examined by the Northern blotting technique usingthe total RNA.

As a result, there were obtained 13 types of clones which did notexpress on 2P15 but expressed on 2P26.

To check the specificity of the cDNAs of the 13 types of clones againstthe human Th2, the difference in expression of mRNA between theplurality of human Th2 clone cells and human Th1 clone cells wasexamined by use of the Northern blotting technique. As a result, therewere obtained several cDNA clones which were observed to have expressionin common for only the human Th2 clone cells. FIG. 1 illustrates theresults of Northern blotting of one (B19) of these clones.

In FIG. 1, it is evident that B19 mRNA has expressed in theaforementioned two human Th2 clones (2P26 and KND4; corresponding tolanes 3 and 4, respectively).

In contrast, B19 mRNA has not expressed in the human Th1 clone (1P04 and2P15; corresponding to lanes 1 and 2, respectively).

The nucleotide sequence of cDNA clone B19 has been analyzed according tothe dideoxy termination method making use of a fluorescent terminator (akit manufactured by Perkin-Elmer was used).

It was revealed that the clone B19 had DNA of a novel nucleotidesequence.

A gene [Th2 (B19) gene of the present invention] containing a nucleotidesequence homologous with the above-described gene of clone B19 wascloned over its entire length.

(4) Cloning of the Th2 (B19) gene

In order to have the full length of the cDNA of interest cloned, a λphage cDNA library was produced from cells which induce a high level ofexpression of B19 mRNA.

Briefly, the total RNA was extracted from the 2P26 cells, and poly(A)⁺RNA was purified by a routine method through use of oligo (dT) latex(Nippon Roche K.K.). Next, double-stranded cDNA was synthesized throughuse of a commercially available cDNA cloning kit (by Life Technologies),and the thus-synthesized cDNA was cloned at the EcoRI site of λ ZAPII(by Stratagene). Subsequently, the in vitro packaging of the λ phageswas completed through use of a commercially available kit (byStratagene). The packaged products were introduced into E. coli XL1-BlueMRF' (by Stratagene), whereby about 1×10⁵ recombinant λ phages wereobtained. The new cDNA fragments obtained in step (3) were labeled with³² P through use of a commercially available random primer labeling kit(by Takara). The labeled fragments, serving as radioactive probes, wereused for the screening of the λ phage library by the plaquehybridization method.

As a result, 42 positive cDNA clones were obtained. Of these positivecDNA clones, three clones having the longest insert DNA were subjectedto the nucleotide sequence analysis performed according to the dideoxytermination method that used a fluorescent terminator similar to thatused in (3). As a result, it was confirmed that nucleotide sequenceslocated in the overlapping areas between the three positive clones ofcDNA completely matched up with one another, and that they had beenderived from the same gene.

Of these three positive clones, the clone B19-1 having the longest cDNAwas named B19 and used in the following procedures.

(5) Structure of the Th2 (B19) gene of the Present Invention

cDNA incorporated into the clone B19 has a strand length of 2911 bp,which is close to the length of the mRNA (about 3 kbp) determined by theNorthern blotting technique. The cDNA has at its 3'-terminal a poly(A)⁺additional signal and ten A's (adenine) which seem to be a part of thepoly(A)⁺.

It was expected that the longest open reading frame would start withATG, the 113th from the 5'-terminal end, and terminate at the 1298th TGAand would encode a protein consisting of 395 amino acid residues. Thenucleotide sequence (CCC ACG ATGT) in the vicinity of the initiationcodon of the gene was similar to the consensus sequence CCA(G) CC ATGGof Kozak (Kozak, M., Nucleic Acids Res., 15, 8125 (1987)).

In view of the foregoing descriptions, the clone B19 was determined tocontain substantially the full length of cDNA which starts from the3'-terminal and reaches up to a part of the untranslated region on the5'-terminal side via the full length of the coding region.

The gene having the cDNA of clone B19 is taken as the Th2 (B19) gene ofthe present invention, and the sequence of this gene is represented bysequence ID No. 5. Further, an amino acid sequence deduced to be encodedby this nucleotide sequence is represented by sequence ID No. 6.

The amino acid sequence is expressed by the single letter representationmethod as follows:

MSANATLKPLCPILEQMSRLQSHSNTSIRYIDHAAVLLHGLASLLGLVENGVILFVVGCRMRQTVVTTWVLHLALSDLLASASLPFFTYFLAVGHSWELGTTFCKLHSSIFFLNMFASGFLLSAISLDRCLQVVRPVWAQNHRTVAAAHKVCLVLWALAVLNTVPYFVFRDTISRLDGRIMCYYNVLLLNPGPDRDATCNSRQAALAVSKFLLAFLVPLAIIASSHAAVSLRLQHRGRRRPGRFVRLVAAVVAAFALCWGPYHVFSLLEARAHANPGLRPLVWRGLPFVTSLAFFNSVANPVLYVLTCPDMLRKLRRSLRTVLESVLVDDSELGGAGSSRRRRTSSTARSASPLALCSRPEEPRGPARLLGWLLGSCAASPQTGPLNRALSSTS

[In the above-described amino acid sequence, A: alanine, V: valine, L:leucine, I: isoleucine, P: proline, F: phenylalanine, W: tryptophan, M:methionine, G: glycine, S: serine, T: threonine, C: cysteine, Q:glutamine, N: asparagine, Y: tyrosine, K: lysine, R: arginine, H:histidine, D: asparatic acid, and E: glutamic acid]

The transformant consisting of E. coli (E. coli K12-JM109 strain) havingthe gene of the present invention integrated therein is deposited withthe National Institute of Bioscience and Human Technology, Agency ofIndustrial Science and Technology, Japan Ministry of International Tradeand Industry (1-3, Tsukuba-shi 1-chome, Ibaraki-ken, Japan 305) underaccetion No. FERM P-15616 (accepted on May 15, 1996).

(6) In vitro Transcription and Translation of Th2 (B19) gene of thePresent Invention

While the Th2 (B19) gene of the present invention was used as atemplate, RNA was synthesized with T7 RNA polymerase through use of acommercially available kit (by Stratagene). Subsequently, the RNA wastranslated in vitro in the presence of ³⁵ S-methionine through use ofcommercially available rabbit reticulocyte extract (by Promega Biotech).

According to the Laemmli method, the thus-translated product wasanalyzed by SDS polyacrylamide gel electrophoresis. As a result, it wasconfirmed that there had been formed a protein having a molecular weightwhich is similar to the predicted molecular weight of 43 kd (see FIG.2).

(7) Tissue Specificity of Expression of mRNA

To examine the tissue specificity of expression of the mRNA derived fromthe Th2 (B19) gene of the present invention, the total RNA of cell linesderived from various tissues was subjected to Northern blot analysis.

As a result, expression of the mRNA derived from the Th2 (B19) genecould not be confirmed in any of the cell lines (FIG. 3).

Thus, it has become evident that expression of the Th2 (B19) gene islimited to a specific cell that includes the Th2 cells.

Example 2

Manufacture of Antibodies Against the Human Th2 (B19) Protein of thePresent Invention

(1) Manufacture of Th2 (B19) Gene Expression Vector

It is difficult to predict the expression level of the introduced genein the mammalian cell because the expression level tends to be widelydifferent dependent on the combination of a host cell and an expressionvector to be used. Therefore, the Th2 (B19) gene was incorporated intosome expression vectors for which high expression was expected.

More specifically, an insert DNA containing the coding region of the Th2(B19) gene was excised from plasmid DNA (pBluescript SK(-), cDNA cloneB19) containing the full length of the Th2 (B19) gene (represented bysequence ID No. 5) through use of restriction enzymes which are suitablefor the cloning sites of the objective expression plasmides. The insertDNA was then purified by agarose gel electrophoresis.

Th2 (B19) gene of the present invention obtained in such a way wasinserted at each of the cloning sites of pRc/CMV, pcDL-SRα, pREP9, etc.,so that various kinds of expression vectors (plasmids) of the Th2 (B19)gene of the present invention for a mammalian cell of interest wereobtained.

(2) Manufacture of Transformed Cell Expressing Th2 (B19) Gene of thePresent Invention

50 μg of each of the human Th2 (B19) protein expression plasmids of thepresent invention obtained in (1) was introduced into 10⁷ cells of eachof Jurkat cells, 293 cells (these being from a human T cell line and ahuman renal cell line), BW 5147 (from a mouse T cell line), and TART-1(from a rat T cell line).

The cells were seeded onto a 96-well plate at 500 to 1000 cells perwell, and cultured in a medium containing Geneticin (by Sigma) for 2 to3 weeks to select transformed cells. The expression level of the Th2(B19) gene of the present invention was confirmed by Northern blottechnique.

The transformed cells having highest expression level of the Th2 (B19)gene of the present invention were selected from a large number of thetransformed cells with respect to each cell line, and then cloningoperation was carried out in which the cells were seeded onto a 96-wellplate at 0.3 cell per well.

As a result, Jurkat/B19, 293/B19, BW/B19 and TART/B19 cell lines whichwere expressing the Th2 (B19) gene of the present invention stably andat a high level were obtained.

Amoung the thus-obtained transformed cells of the Th2 (B19) gene of thepresent invention, 10⁷ cells of the TART/B19 cells were injected into aperitoneal cavity once every week a total of five times to immunize8-week-old Wister rats (female) (Nemoto, T., Eur. J. Immunol., 25, 3001(1995)).

(3) Preparation of Hybridomas

After three days have passed since the rat was finally immunized, 3×10⁸spleen cells of the rat were mixed with 5×10⁷ cells of mouse myelomacell line SP 2/0-Ag14. They were fused together through use of a 50%polyethylene glycol (a mean molecular weight of 1500) PBS solution (bySigma). The fused cells were seeded onto a 96-well plate at 1×10⁵cells/well, and, a HAT reagent (by Sigma) was added from the next dayand the selective culture were carried out for ten days. As a result,the proliferation of hybridomas was observed in substantially all wells.

The presence/absence of monoclonal antibodies specific to the human Th2(B19) protein of the present invention in a culture supernatant of eachhybridoma was confirmed by a membran fluorescent antibody techniqueusing TART-1 cells and TART/B19 cells.

More specificaly, 50 μl of the culture supernatant of each hybridoma wasadded to 5×10⁵ cells of each of TART-1 cells and TART/B19 cells andthoroughly mixed to react each other for 20 min at room temperature.After washing twice with PBS containing 0.5% BSA, the cells were reactedwith phycoerythrin-labeled goat anti-rat immunoglobulin antibody (by BioSource International) for 20 min at room temperature. After thereaction, the cells were washed again twice with PBS containing 0.5% BSAand then floated in a small amount of 50% glycerin/PBS and sealedbetween a slide glass and a cover glass. The fluorescent strength on thecell membranes was observed under a fluorescence microscope (byOlympus).

As a result, the supernetants which had the reactivity to only theTART/B19 cell membrane but not to the TART-1 cell membrane were taken aspositive samples.

(4) Manufacture of Monoclonal Antibodies

With regard to the culture supernatants of the hybridoma which wereadmitted to be positive as a result of the above screening, thespecificity of the supernatants to the human Th2 (B19) protein of thepresent invention was comfirmed with the above plural number of cellpanels.

More specificity, with regard to Jurkat and Jurkat/B109, 293 and293/B19, and BW5147 and BW/B19, the specificity of reaction wasconfirmed by a membrane fluorescent antibody technique as describedabove.

With regard to the hybridomas which were confirmed to have thespecificity in all cell panels as described above, the cloning operationin which the cells were seeded onto a 96-well plate at 0.3 cell per wellwas repeated two or three times, so that the hybridomas which producedmonoclonal antibodies were produced.

One of these hybridomas (Rat Hybridoma BM16) was deposited with theNational Institute of Bioscience and Human Technology, Agency ofIndustrial Science and Technology, Japan Ministry of International Tradeand Industry (1-3, Tsukuba-shi 1-chome, Ibaraki-ken, Japan 305) underaccetion No. FERM P-16216 (accepted on May 8, 1997).

Using this monoclonal antibody, Th2 clone cells were stained by amembrane fluorescent antibody technique as described above. The resultsof analysis by a flow cytometer are shown in FIG. 4.

In FIG. 4, it was confirmed that the product of the Th2 (B19) gene ofthe present invention is expressed on the cell membrane and the productis more dominantly expressed in the Th2 cells than in the Th1 cells.

Industrial Applicability:

By virtue of the present invention, there are provided ahuman-Th2-specific gene and a human-Th2-specific protein which areimportant factors of means for specifying the condition and type ofimmune-related diseases on the basis of the knowledge about thepolarization of distribution of Th1/Th2 subsets.

Further, there are provided a recombinant vector for expressing a genewhich contains the human-Th2-specific gene and a transformant which istransformed by the above-described recombinant vector.

Moreover, there are provided monoclonal and polyclonal antibodies whichuse the human-Th2-specific protein as an antigen, and a hybridoma whichproduces the monoclonal antibody.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 6                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #21                ATGGC T                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #               25 GTAAC TGATA                                                - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #21                GGACT A                                                    - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #               25 CAAGA GCACA                                                - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2911 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #GTCAGGAGCT    60CCACCTC TGTCTGCCCG CTGCCTCTTG TCTAGCTGCT                     #CGATGTCGGC   120GCTGGAA TCCTGTGCTC CCTCTGTGCC CAGAGCCCCA                     #TCCAGAGCCA   180AAGCCAC TCTGCCCCAT CCTGGAGCAG ATGAGCCGTC                     #GGCTGGCCTC   240ATCCGCT ACATCGACCA CGCGGCCGTG CTGCTGCACG                     #GCATGCGCCA   300GTGGAGA ATGGAGTCAT CCTCTTCGTG GTGGGCTGCC                     #CCTCTGCTTC   360ACCTGGG TGCTGCACCT GGCGCTGTCC GACCTGTTGG                     #GCACCACCTT   420ACCTACT TCTTGGCCGT GGGCCACTCG TGGGAGCTGG                     #TCCTGCTCAG   480TCCTCCA TCTTCTTTCT CAACATGTTC GCCAGCGGCT                     #AGAACCACCG   540GACCGCT GCCTGCAGGT GGTGCGGCCG GTGTGGGCGC                     #TGCTCAACAC   600GCGCACA AAGTCTGCCT GGTGCTTTGG GCACTAGCGG                     #TTATGTGCTA   660GTGTTCC GGGACACCAT CTCGCGGCTG GACGGGCGCA                     #ACTCGCGCCA   720CTCCTGA ACCCGGGGCC TGACCGCGAT GCCACGTGCA                     #CGATCATCGC   780GTCAGCA AGTTCCTGCT GGCCTTCCTG GTGCCGCTGG                     #GGCCAGGCCG   840GCCGTGA GCCTGCGGTT GCAGCACCGC GGCCGCCGGC                     #GGCCCTACCA   900GTGGCAG CCGTCGTGGC CGCCTTCGCG CTCTGCTGGG                     #CGCTCGTGTG   960CTGGAGG CGCGGGCGCA CGCAAACCCG GGGCTGCGGC                     #ACCCGGTGCT  1020TTCGTCA CCAGCCTGGC CTTCTTCAAC AGCGTGGCCA                     #GCACGGTGCT  1080TGCCCCG ACATGCTGCG CAAGCTGCGG CGCTCGCTGC                     #GCCGCCGCCG  1140GTGGACG ACAGCGAGCT GGGTGGCGCG GGAAGCAGCC                     #CGGAGGAACC  1200GCCCGCT CGGCCTCCCC TTTAGCTCTC TGCAGCCGCC                     #CCCCGCAGAC  1260CGTCTCC TCGGCTGGCT GCTGGGCAGC TGCGCAGCGT                     #ACGTAGGGCG  1320CGGGCGC TGAGCAGCAC CTCGAGTTAG AACCCGGCCC                     #CGGACTCCTG  1380AAAGTAT CACCAGGGTG CCGCGGTTCA ATTCGATATC                     #GCGCCCCGGG  1440AGTCCGA GGGGCGGGAC CCAGGCACCT GCATTTTAAA                     #CCTTGATGTG  1500TTTTCAG AAACAGTGAG TTAAAGCAGT GCTTCTCAAA                     #GGGGCCGGGT  1560TAGGGGT CTTGTTAAGT GCAGTCTGAT CCAGGAGGCC                     #GGTTCACAGG  1620ACTTAAC AAGCTCCCAG GCCGAGAAGC CAGTGCGGCA                     #GGGCAGTGGA  1680AACACAA AGTGAAACTC GTAATAGACT TCCCACTCTA                     #GATGGGGGAG  1740ACGGGGT GCGTCTCCCC GGAGTTCAGT TTTACCAGAT                     #AGGAAAGGTT  1800TTATGTT AAACCATCCA TGTATTTTTG GAGAAGAGAG                     #GCTAGACGCT  1860TCCAGCC TGCCCTCTTC ATTTAGCCAA TGCTTACTGC                     #AGCACATTCT  1920TTAAGGG GCAGCTTCTA TTAGCCAGTC TTTACAGCTG                     #GGTCTGCACT  1980TAAGTGA CTTGCCCAGT TTCAGGGCTA ACGACCACAG                     #CTCTGACCTA  2040TCACATG CTCAATGACT CTCTGGTGAG CGAGGACATT                     #TCCAAGGCAG  2100AGATGCT ACCTTGTGAC CCAGCACTGC CCAAAGTGCT                     #AAATCCAATG  2160TGGCGTG GTCAAGCACT CGGGAAACCT GGGGCTAATC                     #ACTCTAAGAC  2220AAAGTCT TCGGTCGTTA GAAGTTGAAT GGGCACAGCA                     #GGTGTTCTGT  2280ATTTCTT AGCTAAGCGG ACCAGCCTCC CTGTCGGCCT                     #AGGCCACATG  2340GCACTGG TAATCCCAAG ATCTGTGCAG CCCCGCCTCC                     #CCTCTGACCT  2400ACCATTT CCCTTTTGCG GATGGGAGGG GTAACTTGCA                     #GTCAGAGACT  2460CACCCCG TCTCATTCCT CCACCTGCCG TGGACTTGGG                     #TTTTGCTTGG  2520TCTGCAG CCCAGGGACC GAAAAGTTGG TGTCAATGAA                     #TCCTGTGTTT  2580AGTGGAA GAAGCAGATG AGAAACTCTT GAGATCTTGG                     #CTGAGCAAAG  2640AGGCCAG GGTCACTGAA GGCCTGGCCC ACAGCAGGTG                     #CGCCCCTGCT  2700GCCCAGC TAGCTGCAGA GCCACCCTGT GTTGACACCT                     #TGGTGCATTT  2760TCCCCCT TTACTCATAG CACTTCCCCC ATTGGACACG                     #AGGGACTTTG  2820TGTTTTC TCTCCATCAG AATGAAAGCT CCTCGAGGGC                     #GCACTCAATA  2880ATTTGCC GGTGCCTAGG ATTGTGCCTG TATGCAACAG                     #        2911      GACTG GAAAAAAAAA A                                         - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 395 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #Cys Pro Ile Leu Glu Glnhr Leu Lys Pro Leu                                    #                 15                                                          #Ser Ile Arg Tyr Ile Asper His Ser Asn Thr                                    #             30                                                              #Ser Leu Leu Gly Leu Valeu His Gly Leu Ala                                    #         45                                                                  #Cys Arg Met Arg Gln Threu Phe Val Val Gly                                    #     60                                                                      #Leu Ser Asp Leu Leu Alaal Leu His Leu Ala                                    # 80                                                                          #Leu Ala Val Gly His Serhe Phe Thr Tyr Phe                                    #                 95                                                          #His Ser Ser Ile Phe Phehr Phe Cys Lys Leu                                    #            110                                                              #Ser Ala Ile Ser Leu Asper Gly Phe Leu Leu                                    #        125                                                                  #Ala Gln Asn His Arg Thral Arg Pro Val Trp                                    #    140                                                                      #Leu Trp Ala Leu Ala Valys Val Cys Leu Val                                    #160                                                                          #Asp Thr Ile Ser Arg Leuyr Phe Val Phe Arg                                    #                175                                                          #Leu Leu Leu Asn Pro Glyys Tyr Tyr Asn Val                                    #            190                                                              #Gln Ala Ala Leu Ala Valhr Cys Asn Ser Arg                                    #        205                                                                  #Leu Ala Ile Ile Ala Serla Phe Leu Val Pro                                    #    220                                                                      #His Arg Gly Arg Arg Arger Leu Arg Leu Gln                                    #240                                                                          #Val Val Ala Ala Phe Alarg Leu Val Ala Ala                                    #                255                                                          #Leu Leu Glu Ala Arg Alayr His Val Phe Ser                                    #            270                                                              #Trp Arg Gly Leu Pro Pheeu Arg Pro Leu Val                                    #        285                                                                  #Ala Asn Pro Val Leu Tyrhe Phe Asn Ser Val                                    #    300                                                                      #Leu Arg Arg Ser Leu Argsp Met Leu Arg Lys                                    #320                                                                          #Ser Glu Leu Gly Gly Alaal Leu Val Asp Asp                                    #                335                                                          #Thr Ala Arg Ser Ala Serrg Arg Thr Ser Ser                                    #            350                                                              #Pro Arg Gly Pro Ala Arger Arg Pro Glu Glu                                    #        365                                                                  #Ala Ser Pro Gln Thr Glyeu Gly Ser Cys Ala                                    #    380                                                                      #Serro Leu Asn Arg Ala Leu Ser Ser Thr Ser                                    #395                                                                          __________________________________________________________________________

What is claimed is:
 1. A human-Th2-specific protein comprising an aminoacid sequence set forth in sequence ID No. 6.